OXIDATION OF AN ENGINEERED PORE CYSTEINE LOCKS A VOLTAGE-GATED K+ CHANNEL IN A NONCONDUCTING STATE

We report the use of cysteine-substituted mutants in conjunction with in situ oxidation to determine the physical proximity of a pair of eng ineered cysteines in the pore region of the voltage-gated K+ channel K v2.1. We show that the newly introduced cysteine I379C, located near t he outer end of the narrow ion-conduction pathway, renders the K+ chan nel sensitive to oxidation by H2O2, but only if the native cysteine at position 394 in S6 remains in place. Conservative substitutions in S6 for cysteine 394 abolish H2O2, sensitivity in the Kv2.1 mutant I379C, Comparative immunoblot analysis of wild-type and I379C Kv2.1-expressi ng HEK293 cells demonstrates the presence of subunit dimers for I379C, but not for wild-type Kv2.1, At the single-channel level, the probabi lity of opening of I379C channels, unlike wild-type, is reduced in the presence of H2O2; however, oxidation of I379C does not alter unit cur rent. These findings imply that cysteine 379, located near the outer e nd of the narrow ion-conduction pathway, participates in disulfide bri dge formation, locking the channel in a nonconducting state from which it cannot undergo conformational transitions required for opening.